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Loss-of-co-Homozygosity mapping and exome sequencing of a Syrian pedigree
identified the candidate causal mutation associated with rheumatoid arthritis.
Yukinori Okada1,2, Namrata Gupta2, Daniel Mirel2, Stacey Gabriel2, Thurayya Arayssi3, Faten Mouassess4, Walid AL. Achkar4, Layla A. Kazkaz5,6, Robert M. Plenge1,2.
1. Division of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA. 2. Program in Medical and Population Genetics, Broad
Institute, Cambridge, MA, USA. 3. Weill Cornell Medical College-Qatar, Education City, Doha, Qatar. 4. Molecular Biology and Biotechnology Dept, Human Genetics Division, Damascus,
Syria. 5. Tishreen Hospital, Damascus, Syria. 6. Syrian Association for Rheumatology, Damascus, Syria.
Background/Purpose: Although there are >50 rheumatoid arthritis (RA) risk loci that contain common variants, there are no loci that harbor rare mutations that influence RA risk in a
Mendelian fashion. Here, we perform whole exome sequencing to search for rare, causal mutations in a 4-generation, 49-person consanguineous Syrian pedigree in which 8 individuals
were affected with rheumatoid arthritis (RA).
Method: We performed GWAS genotyping on 16 family members (affected and unaffected) and genome-wide exome sequencing in the 4 anti-CCP positive RA cases. We developed a
novel non-parametric linkage analysis we term “Loss-of-co-Homozygosity” (LOcH) mapping that extends homozygosity mapping to include any type of inheritance mode. LOcH uses
genome-wide SNP data to search the regional stretches that lose one or both homozygous genotypes (i.e., lose “co-homozygosity”) in affected cases, to identify ancestry-shared
haplotype. Candidate mutations selected by exome sequencing and LOcH mapping were further validated by iPlex assay in 24 family members.
Result: Using GWAS data and LOcH mapping, we identified 12% of the genome in which the same ancestral haplotype was shared among all RA cases. Exome sequence identified 15
nonsense or missense candidate mutations shared among all cases. Validation iPlex assay found that 1 mutation preferentially segregated in cases compared to controls (P = 0.023).
The mutated gene is phospholipase B1 (PLB1) at 2p23, which has been implicated in human epidermal barrier function.
Conclusion: While additional investigation of PLB1 mutation is required, our approach highlights a novel method of statistical analysis of genome-wide sequence data.
~ Study design ~
GWAS genotyping of
4 RA cases and 12 controls
~ Syrian family with RA ~
Exome sequence of 4 RA cases
We enrolled consanguineous Syrian
family with rheumatoid arthritis (RA).
49 family members include 8 RA
cases (II-12,13,14, III-3,17,18, IV-5,9) and
1 anti-CCP antibody-positive control
(III-2).
Filtering of nonsense/missense variants
shared among RA cases using
1000Genome/ESP/dbSNP databases
“LOcH mapping” of genetic locus
shared among 4 RA cases
Selection of 15 candidate causal variants
4 RA cases with
▪ Exome sequence,
▪ GWAS genotyping,
▪ iPlex validation assay.
Validation of the candidate variants by iPlex assay
for all available 24 family members
12 controls with
▪ GWAS genotyping,
▪ iPlex validation assay.
4 variants shared among 5 RA cases and
1 anti-CCP antibody positive control
1 RA cases,
1 anti-CCP positive control,
6 controls with
▪ iPlex validation assay.
1 variant at PLB1 gene preferentially segregated
in RA cases compared to controls
~ LOcH (Loss-of-co-Homozygosity) mapping ~
LOcH mapping identifies ancestry-shared
haplotype among affected cases
~ Exome sequence of RA cases ~
LOcH mapping imputes candidate
mutations in non-exomed subjects.
Subjects : 4 RA cases.
Exon capture : Agilent SureSelect Human All Exon Kitv2 (~44Mb).
Sequencer : Illumina HiSeq.
▪ In a family with Mendlian disease,
the causal mutation resides on the
same ancestry-shared haplotype.
▪ Regardless of recessive/dominant
mode of inheritance, all the case
have at least one ancestry-shared
haplotype, which should be “Lossof-co-Homozygosity (LOcH)” in
GWAS data.
▪ LOcH mapping can screen the loci
with the causal mutation, as an
extension of Homozygosity
mapping to a disease with unknown
inheritance mode.
Co-Homozygosity
in genotype counts
AA
AB
BB
+
+
+
AA
AB
BB
+
-
+
Loss-of-co-Homozygosity
in genotype counts
AA
AB
BB
AA
AB
BB
+
-
-
+
+
-
AA
AB
BB
AA
AB
BB
-
+
-
-
+
+
AA
AB
BB
-
-
+
~ LOcH stretches and exome-derived mutations ~
LOcH stretches and SNVs
LOcH stretches and Indels
▪ LOcH mapping can
impute presence of the
exome-derived mutation of
each additional control
using GWAS data in the
LOcH stretch.
▪ When a control has
ancestry-shared haplotype,
LOcH stretch remains
after inclusion of a control.
▪ When a control does not
have ancestry-shared
haplotype, a LOcH stretch
diminishes after inclusion
of a control.
Analysis : GATK pipeline at Broad (GRch37.64).
Mean/Median depth of the variants : 290.1/204.
Genotype concordance with GWAS data : 99.56%
Variant filtering of exome sequence data
65,524 variants identified by exome sequence. Ts/Tv=2.74
14,804 missense / nonsense variants.
900 variants not in dbSNP 132/1000G Phase I/ESP 5400
with non-reference allele frequency ≥0.05.
476/156 SNVs/Indels with available genotypes in all 4 cases.
13/8 SNVs/Indels with ≥1 non-ref alleles in all 4 cases.
~ Candidate causal SNV in PLB1 gene at 2p23 ~
Distribution of PLB1 mutation
▪ We conducted validation iPlex assay of
candidate causal mutations for all 24 available
family subjects.
▪ 13 exome-derived SNVs and 2 Indels included
in LOcH stretches were selected.
▪ 3 SNVs and 1 Indel were observed for all 5 RA
cases and 1 anti-CCP positive control.
▪ Of these, a non-synonymous SNV in
phospholipase B1 (PLB1) gene at 2p23
preferentially segregated in cases compared
to controls (P = 0.023)
Subjects with PLB1 mutation
Subjects without PLB1 mutation
▪ LOcH mapping for 4 RA cases identified 36 LOcH stretches covering 12% of genome.
▪ All exome-derived candidate causal SNVs were included in LOcH stretches (P = 1.1×10-12).
▪ Only 2 of 8 exome-derived candidate causal Indels were included in LOcH stretches (P = 0.44).
▪ JavaTM software for LOcH mapping and genotype imputation is available for the request to the authors.
▪ Distinct overlap rates between SNVs and Indels suggested lower quality of exome-derived indels.
▪ Contact : Yukinori Okada, MD, PhD, [email protected]